Enrichment analysis of phenotypic data for drug repurposing in rare diseases.

Drug-induced Behavioral Signature Analysis (DBSA), is a machine learning (ML) method for in silico screening of compounds, inspired by analytical methods quantifying gene enrichment in genomic analyses. When applied to behavioral data it can identify drugs that can potentially reverse in vivo behavioral symptoms in animal models of human disease and suggest new hypotheses for drug discovery and repurposing. We present a proof-of-concept study aiming to assess Drug-induced Behavioral Signature Analysis (DBSA) as a systematic approach for drug discovery for rare disorders. We applied Drug-induced Behavioral Signature Analysis to high-content behavioral data obtained with SmartCube®, an automated in vivo phenotyping platform. The therapeutic potential of several dozen approved drugs was assessed for phenotypic reversal of the behavioral profile of a Huntington's Disease (HD) murine model, the Q175 heterozygous knock-in mice. The in silico Drug-induced Behavioral Signature Analysis predictions were enriched for drugs known to be effective in the symptomatic treatment of Huntington's Disease, including bupropion, modafinil, methylphenidate, and several SSRIs, as well as the atypical antidepressant tianeptine. To validate the method, we tested acute and chronic effects of tianeptine (20 mg/kg, i. p.) in vivo, using Q175 mice and wild type controls. In both experiments, tianeptine significantly rescued the behavioral phenotype assessed with the SmartCube® platform. Our target-agnostic method thus showed promise for identification of symptomatic relief treatments for rare disorders, providing an alternative method for hypothesis generation and drug discovery for disorders with huge disease burden and unmet medical needs.

Benefits of global mutant huntingtin lowering diminish over time in a Huntington's disease mouse model.

We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.

Computational Analysis of Multidimensional Behavioral Alterations After Chronic Social Defeat Stress.

BACKGROUND: The study of depression in humans depends on animal models that attempt to mimic specific features of the human syndrome. Most studies focus on one or a few behavioral domains, with time and practical considerations prohibiting a comprehensive evaluation. Although machine learning has enabled unbiased analysis of behavior in animals, this has not yet been applied to animal models of psychiatric disease. METHODS: We performed chronic social defeat stress (CSDS) in mice and evaluated behavior with PsychoGenics' SmartCube, a high-throughput unbiased automated phenotyping platform that collects >2000 behavioral features based on machine learning. We evaluated group differences at several times post-CSDS and after administration of the antidepressant medication imipramine. RESULTS: SmartCube analysis after CSDS successfully separated control and defeated-susceptible mice, and defeated-resilient mice more resembled control mice. We observed a potentiation of CSDS effects over time. Treatment of susceptible mice with imipramine induced a 40.2% recovery of the defeated-susceptible phenotype as assessed by SmartCube. CONCLUSIONS: High-throughput analysis can simultaneously evaluate multiple behavioral alterations in an animal model for the study of depression, which provides a more unbiased and holistic approach to evaluating group differences after CSDS and perhaps can be applied to other mouse models of psychiatric disease.

Combinatorial ETS1-dependent control of oncogenic NOTCH1 enhancers in T-cell leukemia.

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to co-bind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis, but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and points to Ets1-mediated enucleation of Notch-Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition.

Mutational and functional genetics mapping of chemotherapy resistance mechanisms in relapsed acute lymphoblastic leukemia.

Multi-agent combination chemotherapy can be curative in acute lymphoblastic leukemia (ALL). Still, patients with primary refractory disease or with relapsed leukemia have a very poor prognosis. Here we integrate an in-depth dissection of the mutational landscape across diagnostic and relapsed pediatric and adult ALL samples with genome-wide CRISPR screen analysis of gene-drug interactions across seven ALL chemotherapy drugs. By combining these analyses, we uncover diagnostic and relapse-specific mutational mechanisms as well as genetic drivers of chemoresistance. Functionally, our data identifies common and drug-specific pathways modulating chemotherapy response and underscores the effect of drug combinations in restricting the selection of resistance-driving genetic lesions. In addition, by identifying actionable targets for the reversal of chemotherapy resistance, these analyses open novel therapeutic opportunities for the treatment of relapse and refractory disease.

Phf6 Loss Enhances HSC Self-Renewal Driving Tumor Initiation and Leukemia Stem Cell Activity in T-ALL.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. SIGNIFICANCE: Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL.This article is highlighted in the In This Issue feature, p. 305.

Oncogenic hijacking of the stress response machinery in T cell acute lymphoblastic leukemia.

Cellular transformation is accompanied by extensive rewiring of many biological processes leading to augmented levels of distinct types of cellular stress, including proteotoxic stress. Cancer cells critically depend on stress-relief pathways for their survival. However, the mechanisms underlying the transcriptional initiation and maintenance of the oncogenic stress response remain elusive. Here, we show that the expression of heat shock transcription factor 1 (HSF1) and the downstream mediators of the heat shock response is transcriptionally upregulated in T cell acute lymphoblastic leukemia (T-ALL). Hsf1 ablation suppresses the growth of human T-ALL and eradicates leukemia in mouse models of T-ALL, while sparing normal hematopoiesis. HSF1 drives a compact transcriptional program and among the direct HSF1 targets, specific chaperones and co-chaperones mediate its critical role in T-ALL. Notably, we demonstrate that the central T-ALL oncogene NOTCH1 hijacks the cellular stress response machinery by inducing the expression of HSF1 and its downstream effectors. The NOTCH1 signaling status controls the levels of chaperone/co-chaperone complexes and predicts the response of T-ALL patient samples to HSP90 inhibition. Our data demonstrate an integral crosstalk between mediators of oncogene and non-oncogene addiction and reveal critical nodes of the heat shock response pathway that can be targeted therapeutically.

Structure and Mechanisms of NT5C2 Mutations Driving Thiopurine Resistance in Relapsed Lymphoblastic Leukemia.

Activating mutations in the cytosolic 5'-nucleotidase II gene NT5C2 drive resistance to 6-mercaptopurine in acute lymphoblastic leukemia. Here we demonstrate that constitutively active NT5C2 mutations K359Q and L375F reconfigure the catalytic center for substrate access and catalysis in the absence of allosteric activator. In contrast, most relapse-associated mutations, which involve the arm segment and residues along the surface of the inter-monomeric cavity, disrupt a built-in switch-off mechanism responsible for turning off NT5C2. In addition, we show that the C-terminal acidic tail lost in the Q523X mutation functions to restrain NT5C2 activation. These results uncover dynamic mechanisms of enzyme regulation targeted by chemotherapy resistance-driving NT5C2 mutations, with important implications for the development of NT5C2 inhibitor therapies.

RHOA G17V Induces T Follicular Helper Cell Specification and Promotes Lymphomagenesis.

Angioimmunoblastic T cell lymphoma (AITL) is an aggressive tumor derived from malignant transformation of T follicular helper (Tfh) cells. AITL is characterized by loss-of-function mutations in Ten-Eleven Translocation 2 (TET2) epigenetic tumor suppressor and a highly recurrent mutation (p.Gly17Val) in the RHOA small GTPase. Yet, the specific role of RHOA G17V in AITL remains unknown. Expression of Rhoa G17V in CD4+ T cells induces Tfh cell specification; increased proliferation associated with inducible co-stimulator (ICOS) upregulation and increased phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase signaling. Moreover, RHOA G17V expression together with Tet2 loss resulted in development of AITL in mice. Importantly, Tet2-/-RHOA G17V tumor proliferation in vivo can be inhibited by ICOS/PI3K-specific blockade, supporting a driving role for ICOS signaling in Tfh cell transformation.

Clonal evolution mechanisms in NT5C2 mutant-relapsed acute lymphoblastic leukaemia.

Relapsed acute lymphoblastic leukaemia (ALL) is associated with resistance to chemotherapy and poor prognosis. Gain-of-function mutations in the 5'-nucleotidase, cytosolic II (NT5C2) gene induce resistance to 6-mercaptopurine and are selectively present in relapsed ALL. Yet, the mechanisms involved in NT5C2 mutation-driven clonal evolution during the initiation of leukaemia, disease progression and relapse remain unknown. Here we use a conditional-and-inducible leukaemia model to demonstrate that expression of NT5C2(R367Q), a highly prevalent relapsed-ALL NT5C2 mutation, induces resistance to chemotherapy with 6-mercaptopurine at the cost of impaired leukaemia cell growth and leukaemia-initiating cell activity. The loss-of-fitness phenotype of NT5C2+/R367Q mutant cells is associated with excess export of purines to the extracellular space and depletion of the intracellular purine-nucleotide pool. Consequently, blocking guanosine synthesis by inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) induced increased cytotoxicity against NT5C2-mutant leukaemia lymphoblasts. These results identify the fitness cost of NT5C2 mutation and resistance to chemotherapy as key evolutionary drivers that shape clonal evolution in relapsed ALL and support a role for IMPDH inhibition in the treatment of ALL.

WT1 loss attenuates the TP53-induced DNA damage response in T-cell acute lymphoblastic leukemia.

Loss-of-function mutations and deletions in Wilms tumor 1 (WT1) gene are present in approximately 10% of T-cell acute lymphoblastic leukemia. Clinically, WT1 mutations are enriched in relapsed series and are associated to inferior relapse-free survival in thymic T-cell acute lymphoblastic leukemia cases. Here we demonstrate that WT1 plays a critical role in the response to DNA damage in T-cell leukemia. WT1 loss conferred resistance to DNA damaging agents and attenuated the transcriptional activation of important apoptotic regulators downstream of TP53 in TP53-competent MOLT4 T-leukemia cells but not in TP53-mutant T-cell acute lymphoblastic leukemia cell lines. Notably, WT1 loss positively affected the expression of the X-linked inhibitor of apoptosis protein, XIAP, and genetic or chemical inhibition with embelin (a XIAP inhibitor) significantly restored sensitivity to γ-radiation in both T-cell acute lymphoblastic leukemia cell lines and patient-derived xenografts. These results reveal an important role for the WT1 tumor suppressor gene in the response to DNA damage, and support the view that anti-XIAP targeted therapies could have a role in the treatment of WT1-mutant T-cell leukemia.

Pharmacological inhibition of the transcription factor PU.1 in leukemia.

The transcription factor PU.1 is often impaired in patients with acute myeloid leukemia (AML). Here, we used AML cells that already had low PU.1 levels and further inhibited PU.1 using either RNA interference or, to our knowledge, first-in-class small-molecule inhibitors of PU.1 that we developed specifically to allosterically interfere with PU.1-chromatin binding through interaction with the DNA minor groove that flanks PU.1-binding motifs. These small molecules of the heterocyclic diamidine family disrupted the interaction of PU.1 with target gene promoters and led to downregulation of canonical PU.1 transcriptional targets. shRNA or small-molecule inhibition of PU.1 in AML cells from either PU.1lo mutant mice or human patients with AML-inhibited cell growth and clonogenicity and induced apoptosis. In murine and human AML (xeno)transplantation models, treatment with our PU.1 inhibitors decreased tumor burden and resulted in increased survival. Thus, our study provides proof of concept that PU.1 inhibition has potential as a therapeutic strategy for the treatment of AML and for the development of small-molecule inhibitors of PU.1.

Synergistic antileukemic therapies in NOTCH1-induced T-ALL.

The Notch1 gene is a major oncogenic driver and therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL). However, inhibition of NOTCH signaling with γ-secretase inhibitors (GSIs) has shown limited antileukemic activity in clinical trials. Here we performed an expression-based virtual screening to identify highly active antileukemic drugs that synergize with NOTCH1 inhibition in T-ALL. Among these, withaferin A demonstrated the strongest cytotoxic and GSI-synergistic antileukemic effects in vitro and in vivo. Mechanistically, network perturbation analyses showed eIF2A-phosphorylation-mediated inhibition of protein translation as a critical mediator of the antileukemic effects of withaferin A and its interaction with NOTCH1 inhibition. Overall, these results support a role for anti-NOTCH1 therapies and protein translation inhibitor combinations in the treatment of T-ALL.

A case study of an integrative genomic and experimental therapeutic approach for rare tumors: identification of vulnerabilities in a pediatric poorly differentiated carcinoma.

BACKGROUND: Precision medicine approaches are ideally suited for rare tumors where comprehensive characterization may have diagnostic, prognostic, and therapeutic value. We describe the clinical case and molecular characterization of an adolescent with metastatic poorly differentiated carcinoma (PDC). Given the rarity and poor prognosis associated with PDC in children, we utilized genomic analysis and preclinical models to validate oncogenic drivers and identify molecular vulnerabilities. METHODS: We utilized whole exome sequencing (WES) and transcriptome analysis to identify germline and somatic alterations in the patient's tumor. In silico and in vitro studies were used to determine the functional consequences of genomic alterations. Primary tumor was used to generate a patient-derived xenograft (PDX) model, which was used for in vivo assessment of predicted therapeutic options. RESULTS: WES revealed a novel germline frameshift variant (p.E1554fs) in APC, establishing a diagnosis of Gardner syndrome, along with a somatic nonsense (p.R790*) APC mutation in the tumor. Somatic mutations in TP53, MAX, BRAF, ROS1, and RPTOR were also identified and transcriptome and immunohistochemical analyses suggested hyperactivation of the Wnt/ß-catenin and AKT/mTOR pathways. In silico and biochemical assays demonstrated that the MAX p.R60Q and BRAF p.K483E mutations were activating mutations, whereas the ROS1 and RPTOR mutations were of lower utility for therapeutic targeting. Utilizing a patient-specific PDX model, we demonstrated in vivo activity of mTOR inhibition with temsirolimus and partial response to inhibition of MEK. CONCLUSIONS: This clinical case illustrates the depth of investigation necessary to fully characterize the functional significance of the breadth of alterations identified through genomic analysis.

Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia.

Although multiagent combination chemotherapy is curative in a significant fraction of childhood acute lymphoblastic leukemia (ALL) patients, 20% of cases relapse and most die because of chemorefractory disease. Here we used whole-exome and whole-genome sequencing to analyze the mutational landscape at relapse in pediatric ALL cases. These analyses identified numerous relapse-associated mutated genes intertwined in chemotherapy resistance-related protein complexes. In this context, RAS-MAPK pathway-activating mutations in the neuroblastoma RAS viral oncogene homolog (NRAS), kirsten rat sarcoma viral oncogene homolog (KRAS), and protein tyrosine phosphatase, nonreceptor type 11 (PTPN11) genes were present in 24 of 55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, whereas in others RAS mutant clones present at diagnosis were replaced by RAS wild-type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Consistently, functional dissection of mouse and human wild-type and mutant RAS isogenic leukemia cells demonstrated induction of methotrexate resistance but also improved the response to vincristine in mutant RAS-expressing lymphoblasts. These results highlight the central role of chemotherapy-driven selection as a central mechanism of leukemia clonal evolution in relapsed ALL, and demonstrate a previously unrecognized dual role of RAS mutations as drivers of both sensitivity and resistance to chemotherapy.

Metabolic reprogramming induces resistance to anti-NOTCH1 therapies in T cell acute lymphoblastic leukemia.

Activating mutations in NOTCH1 are common in T cell acute lymphoblastic leukemia (T-ALL). Here we identify glutaminolysis as a critical pathway for leukemia cell growth downstream of NOTCH1 and a key determinant of the response to anti-NOTCH1 therapies in vivo. Mechanistically, inhibition of NOTCH1 signaling in T-ALL induces a metabolic shutdown, with prominent inhibition of glutaminolysis and triggers autophagy as a salvage pathway supporting leukemia cell metabolism. Consequently, inhibition of glutaminolysis and inhibition of autophagy strongly and synergistically enhance the antileukemic effects of anti-NOTCH1 therapy in mice harboring T-ALL. Moreover, we demonstrate that Pten loss upregulates glycolysis and consequently rescues leukemic cell metabolism, thereby abrogating the antileukemic effects of NOTCH1 inhibition. Overall, these results identify glutaminolysis as a major node in cancer metabolism controlled by NOTCH1 and as therapeutic target for the treatment of T-ALL.

Therapeutic targeting of HES1 transcriptional programs in T-ALL.

Oncogenic activation of NOTCH1 signaling plays a central role in the pathogenesis of T-cell acute lymphoblastic leukemia, with mutations on this signaling pathway affecting more than 60% of patients at diagnosis. However, the transcriptional regulatory circuitries driving T-cell transformation downstream of NOTCH1 remain incompletely understood. Here we identify Hairy and Enhancer of Split 1 (HES1), a transcriptional repressor controlled by NOTCH1, as a critical mediator of NOTCH1-induced leukemogenesis strictly required for tumor cell survival. Mechanistically, we demonstrate that HES1 directly downregulates the expression of BBC3, the gene encoding the PUMA BH3-only proapoptotic factor in T-cell acute lymphoblastic leukemia. Finally, we identify perhexiline, a small-molecule inhibitor of mitochondrial carnitine palmitoyltransferase-1, as a HES1-signature antagonist drug with robust antileukemic activity against NOTCH1-induced leukemias in vitro and in vivo.

Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks.

We introduce a method for simultaneous prediction of microRNA-target interactions and their mediated competitive endogenous RNA (ceRNA) interactions. Using high-throughput validation assays in breast cancer cell lines, we show that our integrative approach significantly improves on microRNA-target prediction accuracy as assessed by both mRNA and protein level measurements. Our biochemical assays support nearly 500 microRNA-target interactions with evidence for regulation in breast cancer tumors. Moreover, these assays constitute the most extensive validation platform for computationally inferred networks of microRNA-target interactions in breast cancer tumors, providing a useful benchmark to ascertain future improvements.

A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia.

Efforts to identify and annotate cancer driver genetic lesions have been focused primarily on the analysis of protein-coding genes; however, most genetic abnormalities found in human cancer are located in intergenic regions. Here we identify a new long range-acting MYC enhancer controlled by NOTCH1 that is targeted by recurrent chromosomal duplications in human T cell acute lymphoblastic leukemia (T-ALL). This highly conserved regulatory element, hereby named N-Me for NOTCH MYC enhancer, is located within a broad super-enhancer region +1.47 Mb from the MYC transcription initiating site, interacts with the MYC proximal promoter and induces orientation-independent MYC expression in reporter assays. Moreover, analysis of N-Me knockout mice demonstrates a selective and essential role of this regulatory element during thymocyte development and in NOTCH1-induced T-ALL. Together these results identify N-Me as a long-range oncogenic enhancer implicated directly in the pathogenesis of human leukemia and highlight the importance of the NOTCH1-MYC regulatory axis in T cell transformation and as a therapeutic target in T-ALL.

Small Molecule that Reverses Dexamethasone Resistance in T-cell Acute Lymphoblastic Leukemia (T-ALL).

Glucocorticoids are one of the most utilized and effective therapies in treating T-cell acute lymphoblastic leukemia. However, patients often develop resistance to glucocorticoids, rendering these therapies ineffective. We screened 9517 compounds, selected for their lead-like properties, chosen from among 3 372 615 compounds, against a dexamethasone-resistant T-ALL cell line to identify small molecules that reverse glucocorticoid resistance. We synthesized analogues of the most effective compound, termed J9, from the screen in order to define the scaffold's structure-activity relationship. Active compounds restored sensitivity to glucocorticoids through upregulation of the glucocorticoid receptor. This compound and mechanism may provide a strategy for overcoming glucocorticoid resistance in patients with T-ALL.